The broad, long-term objective of this project is to capitalize on the strengths of the P. knowlesi in vivo non- human primate model system to identify, distinguish and characterize the molecular mechanisms that regulate antigenic variation in Plasmodium. Antigenic variation in Plasmodium is a fundamental adaptation to evade a host protective immune response and one of the major factors contributing to the establishment of chronic malaria blood infections and severe pathology. As Plasmodium falciparum develops in an infected erythrocyte it produces high molecular weight proteins that become exposed at the surface of these host cells. These protein antigens vary in the course of an immune response by switching the expression of over 50 members of a large gene family, called the SICAvar gene family in P. knowlesi and the var gene family in P. falciparum. The precise genetic and immunobiological mechanisms regulating this process in vivo remain largely unknown, and, to date, speculative at best. The P. knowlesi simian malaria/rhesus monkey model - originally used to discover antigenic variation in malaria - uniquely enables investigations of the mechanisms that operate in vivo as well as in vitro at the DMA, RNA and protein levels as changes in phenotypic expression occur. We have demonstrated using proteomic technologies that the surface exposed variant antigens of P. knowlesi, known as the SICA (Schizont Infected Cell Agglutination) antigens, are related to the P. falciparum variant antigens called the Erythrocyte Membrane Protein-1 (EMP1), corroborating earlier biological data in support of these relationships. Importantly, the P. knowlesi model enables the development and investigation of in vivo derived, isogenic related clones exhibiting distinct stable SICA[+] phenotypes;as well as SICA[-] phenotypes after passage in splenectomized animals. These cloned P. knowlesi parasite populations allow for carefully controlled studies related to variant antigen expression, switching, and the in vivo interplay with the host immune response. Using this model system we identified the SICA var gene family and determined that the expression of the 205 kDa SICA antigen in Pk1(B+)1 + parasites is the result of a specific recombination event involving conserved yet polymorphic 3'coding and non-coding sequences. The specific aims of this new project relating to the investigations of mechanisms that regulate antigenic variation in Plasmodium are to: 1) Investigate the hypothesis that specific 3'genomic DNA recombination events, as observed to date, are associated with in vivo switching and activation of SICA var genes, resulting in their transcription and translation, and 2) Investigate RNA characteristics and putative functional traits of expressed and non-expressed SICAvar genes in up to three stable isogenic SICA[+] and SICA[-] clonal parasite lines derived from successive switching events.